CN115244402A

CN115244402A - Method for measuring blood coagulation time

Info

Publication number: CN115244402A
Application number: CN202180019335.6A
Authority: CN
Inventors: 川边俊树
Original assignee: Sekisui Medical Co Ltd
Current assignee: Sekisui Medical Co Ltd
Priority date: 2020-03-06
Filing date: 2021-03-05
Publication date: 2022-10-25
Also published as: EP4116714A1; EP4116714A4; JPWO2021177452A1; US20230152335A1; WO2021177452A1

Abstract

The invention provides a method for accurately measuring the coagulation time of blood samples showing various coagulation reactions. A blood clotting time measuring method comprising: measuring a coagulation reaction of a sample obtained by mixing a sample to be measured and a reagent for measuring a coagulation time; calculating a weighted average time of a region to be operated with respect to a waveform relating to a coagulation velocity from the obtained measurement data; and determining the weighted average time as the blood coagulation time.

Description

Method for measuring blood coagulation time

Technical Field

The invention relates to a method for measuring blood coagulation time.

Background

The blood coagulation test is a test for diagnosing the blood coagulation ability of a patient by adding a predetermined reagent to a blood sample of the patient to measure the blood coagulation time or the like. Typical examples of the blood coagulation time include Prothrombin Time (PT), activated Partial Thromboplastin Time (APTT), and thrombin time. The hemostatic ability and fibrinolytic ability of a patient can be investigated by blood coagulation test. Abnormalities in the clotting capacity of blood mainly result in prolonged clotting times. For example, the prolongation of the coagulation time is caused by the influence of an anticoagulant, the decrease in components involved in coagulation, the deficiency of an innate blood coagulation factor, an acquired autoantibody that inhibits the coagulation reaction, and the like.

In recent years, automatic analyzers that perform automatic measurement of blood coagulation tests have been widely used, and blood coagulation tests can be easily performed. For example, in some automatic analyzers, a mixed solution obtained by adding a reagent to a blood sample is irradiated with light, and the coagulation reaction of the blood sample is measured based on the change in the amount of the light obtained. For example, in the case of measuring the amount of scattered light, the amount of scattered light is rapidly increased by the progress of coagulation when a certain amount of time has elapsed from the start of adding a reagent to a blood sample, and thereafter, the coagulation reaction is nearly completed and the amount of scattered light is saturated and stabilized. The blood coagulation time can be measured based on the change with time of the amount of scattered light.

As a method for calculating the coagulation time by an automatic analyzer, several methods such as a percentage method and a differential method are used (see patent document 1). In the case of calculating the coagulation time based on the amount of scattered light, in the percentage method, typically, the time until the measured amount of scattered light reaches a certain ratio of the maximum value thereof is calculated as the coagulation time. The percentage method can be used for calculating the coagulation time of normal samples and abnormal samples such as low-fibrinogen samples, chyle samples, hemolytic samples and the like quite accurately. On the other hand, in the automatic analysis by the percentage method, it is necessary to set the measurement time of the sample to be long so that the maximum amount of scattered light can be detected even with an abnormal sample having a low coagulability such as a low fibrinogen sample, and therefore, it takes time to analyze.

In the differentiation method, typically, the coagulation time is calculated as the time taken until the differential value of the scattered light amount reaches a peak or a certain ratio thereof. However, when an abnormal sample having a low coagulability such as a low fibrinogen sample is used, a clear peak may not be observed in the differential value of the scattered light amount. In the abnormal sample, peaks of 2 or more differential values may occur. In some cases, a method of calculating the coagulation time based on a unimodal curve created by fitting a differential value curve is used, and the fitting may impair accurate information on the coagulation ability of the sample.

Further, photometric data in the analyzer includes various noises caused by the state of the device, the reagent, and the sample, and these noises may cause erroneous detection of the coagulation time. For automatic analysis of blood samples, it is required to calculate a reliable coagulation time by removing the adverse effect of noise.

Documents of the prior art

Patent document

Patent document 1: japanese patent laid-open publication No. 6-249855

Disclosure of Invention

The present invention relates to a method for measuring blood coagulation time, which can accurately measure the coagulation time of blood samples showing various blood coagulation reaction curves.

Namely, the present invention provides the following method.

[ 1] A method for measuring blood coagulation time, comprising:

measuring a coagulation reaction of a sample obtained by mixing a sample to be measured and a reagent for measuring a coagulation time,

calculating a weighted average time of the region to be operated with respect to the waveform relating to the coagulation velocity from the obtained measurement data, an

Determining a blood clotting time of the measured sample based on the weighted average time;

the calculation target region is a region in which the waveform of the waveform relating to the coagulation speed is equal to or greater than a predetermined lower limit value.

The method according to [ 1], wherein the coagulation rate-related waveform is a coagulation response curve or a first order differential curve of relative values thereof.

[ 3] the method according to [ 1] or [ 2], wherein the weighted average time is represented by the following formula when the waveform relating to the coagulation speed is F (t) (t is time) and the time when F (t) is x% of the maximum value (x is a predetermined value set in a range of 5 to 95) is t1 and t2 (t 1 < t 2):

the method according to any one of [ 1] to [ 3], wherein the sample to be measured is plasma.

The method according to any one of [ 1] to [ 4 ], wherein the blood coagulation time is Activated Partial Thromboplastin Time (APTT), prothrombin Time (PT), or coagulation time in measurement of fibrinogen concentration.

[ 6 ] A method for measuring a coagulation factor concentration, which comprises measuring the coagulation factor concentration of a sample to be measured based on the blood coagulation time of the sample measured by any one of the methods [ 1] to [ 5 ].

The method according to [ 6 ], wherein the sample to be tested is plasma.

The method according to [ 6 ] or [ 7 ], wherein the coagulation factor is fibrinogen.

The method according to [ 9 ] or [ 8 ], wherein the blood coagulation time is a coagulation time in measurement of fibrinogen concentration.

According to the method of the present invention, the coagulation time of a blood sample showing various blood coagulation response curves including a normal sample and an abnormal sample can be accurately measured. Further, when a large number of samples are analyzed in real time by an automatic analyzer, the method of the present invention can shorten the analysis time per 1 sample as compared with the conventional percentage method, and can improve the analysis efficiency.

Drawings

FIG. 1 is a basic flow chart of one embodiment of the blood coagulation time measuring method of the present invention.

Fig. 2 is an embodiment of the sequence of the data analysis process shown in fig. 1.

FIG. 3 is an example of a coagulation response curve.

FIG. 4 is an example of a coagulation response curve after pretreatment.

FIG. 5 is A: partial enlargement of an example of the solidification reaction curve, B: an enlarged view of a portion of an example of a coagulation response curve after pretreatment.

Fig. 6 is an example of correcting the 0 th order curve.

Fig. 7 is an example of a calibration first order curve.

Fig. 8 is a conceptual diagram showing the calculation target region and the weighted average point.

Fig. 9 is a conceptual diagram showing a change in weighted average point according to the operation target region.

Fig. 10 is a linear regression line of vT20% in the 20% operation target region with respect to APTT by the percentage method.

Fig. 11 shows the slope (a), intercept (B), and correlation coefficient (C) of the linear regression line with respect to the APTT by the percentage method for vT (vT 5% to vT 95%) in 5 to 95% of the operation target region.

Fig. 12 shows the error of the control (APTT by percentage method) with respect to the weighted average time (vT 5% to vT 95%) in 5 to 95% of the calculation target region for 24 samples. The gray units in the table represent the difference between the weighted average time and the control, which represents within + -5% (A) and within + -2.5% (B) of the control.

Fig. 13 shows a linear regression line of vT30% in the 30% operation target region with respect to PT by the percentage method.

Fig. 14 shows the slope (a), intercept (B), and correlation coefficient (C) of the linear regression line with respect to PT by the percentage method for vT (vT 5% to vT 95%) in 5 to 95% of the calculation target region.

Fig. 15 shows the error of the weighted average time (vT 5% to vT 95%) in 5 to 95% of the calculation target regions for 23 samples with respect to the control (PT by the percentage method). The gray units in the table indicate that the difference between the weighted average time and the control is within + -5% (A) and within + -2.5% (B) of the control.

Fig. 16 is a linear regression line of the [ Fbg ] calculated value by the weighted average time with respect to the [ Fbg ] calculated value by the percentage method in the 35% operation target region.

Fig. 17 shows the slope (a), intercept (B), and correlation coefficient (C) of a linear regression line with respect to the [ Fbg ] calculated value by the percentage method, based on the [ Fbg ] calculated value of vT (vT 5% to vT 95%) in the 5 to 95% calculation target region.

Fig. 18 shows an error of the calculated value of [ Fbg ] with respect to the expected value in 5 to 95% of the calculation target region of the density series sample data of 20 samples. The gray units in the table indicate that the error between the calculated value and the expected value is within ± 10% (a) and within ± 5% (B).

Detailed Description

In the blood coagulation test, a predetermined reagent is added to a blood sample, the subsequent blood coagulation reaction is measured, and the blood coagulation time is measured from the coagulation reaction. In the present specification, a blood sample is sometimes referred to as a sample. The measurement of the blood coagulation reaction can be performed by a general means such as an optical means for measuring the amount of scattered light, transmittance, absorbance, or the like, or a mechanical means for measuring the viscosity of plasma. The coagulation response curve of a normal sample depends on the measurement means, but basically shows an S-shape. For example, a coagulation reaction curve based on the amount of scattered light of a normal sample generally rises sharply due to progress of coagulation when a certain amount of time has elapsed since the addition of a reagent, and thereafter, the coagulation reaction approaches the end and becomes smooth. On the other hand, the coagulation response curve of an abnormal sample having coagulation abnormality shows various shapes due to abnormality such as delay in the rise time of the curve, gradual rise, and the like. The coagulation reaction curves of various abnormal samples are not easy to be measured accurately by an automatic analyzer.

In a conventional general blood coagulation time measurement, data at least up to the end of a coagulation reaction is acquired, and a coagulation time is calculated based on the acquired data. For example, when the coagulation time is calculated based on the amount of scattered light, a method (differential method) in which the coagulation reaction curve reaches the maximum speed or 1/N thereof during the period from the time when the reagent is added to the time when the coagulation reaction is completed after the time when the amount of scattered light is saturated is determined as the coagulation reaction is completed, and a method (percentage method, see patent document 1) in which the coagulation time is determined when the amount of scattered light reaches 1/N of the time when the coagulation reaction is completed are known. However, the abnormal shape or noise of the coagulation reaction curve of the abnormal sample may cause a peak of the coagulation reaction rate or erroneous detection of the completion of the coagulation reaction, and the detection may be performed at a time point when the reaction rate peak or the reaction completion is early. Such false detection can result in an incorrect coagulation time being calculated.

In order to efficiently analyze a large number of samples, it is desirable for an automatic analyzer to acquire necessary data for one sample, then promptly terminate measurement, and then start measurement of the next sample. However, in this method, the false detection of the end of the coagulation reaction at the aforementioned premature moment leads to premature measurement ending, with the risk of losing the required data. On the other hand, if the coagulation reaction measurement time of each sample is fixed to a sufficiently long time, data loss caused by erroneous detection of the end of the coagulation reaction can be prevented. However, this method unnecessarily lengthens the measurement time for a large number of samples, thus reducing the overall analysis efficiency.

The present invention can prevent erroneous detection of the coagulation time due to the abnormal shape of the coagulation reaction curve and measure the correct coagulation time. Further, according to the present invention, since the minimum coagulation reaction measurement time required for each coagulation time measurement can be applied to various blood samples including normal samples and abnormal samples, the analysis time per 1 sample can be shortened.

[ method of measuring blood coagulation time ]

The invention relates to a method for measuring blood coagulation time of a blood sample. The method for measuring blood coagulation time of the present invention (hereinafter, also referred to as the method of the present invention) includes: measuring a coagulation reaction of a sample obtained by mixing a sample to be measured and a reagent for measuring a coagulation time; from the obtained measurement data, a weighted average time of a predetermined calculation target region of a waveform relating to the coagulation speed is calculated. One embodiment of the method of the present invention is illustrated with reference to fig. 1. In the method, a sample is first prepared from a sample to be measured, and then a measurement of a coagulation reaction is performed with respect to the sample (step 1). The obtained measurement data is analyzed to obtain a waveform relating to the coagulation rate of the sample, and then a weighted average time of a predetermined region to be calculated relating to the waveform is calculated (step 2). Based on the obtained weighted average time, the coagulation time of the sample to be measured is determined (step 3).

1. Measurement of coagulation reaction

In the measurement of the coagulation reaction, the coagulation reaction of a sample to be measured in which reagents are mixed is measured. The blood coagulation time is determined based on the time-series data of the coagulation reaction obtained by the measurement. Examples of the blood coagulation time measured by the method of the present invention include a Prothrombin Time (PT), an Activated Partial Thromboplastin Time (APTT), a coagulation time in measurement of a fibrinogen concentration (Fbg), and the like. Hereinafter, the method of the present invention will be described by mainly taking an Activated Partial Thromboplastin Time (APTT) as a coagulation time. The method of the present invention may be modified for other coagulation times (e.g., for Prothrombin Time (PT)), as long as the method is performed by those skilled in the art.

In the method of the present invention, as the blood sample to be measured, plasma of the subject can be preferably used. An anti-coagulating agent generally used in coagulation test may be added to the sample. For example, blood is collected using a blood collection tube containing sodium citrate, and then plasma is obtained by centrifugation.

A reagent for measuring clotting time is added to the sample to be measured, and a blood clotting reaction is started. The coagulation reaction of the mixed solution after the reagent is added can be measured. The reagent for measuring clotting time used can be arbitrarily selected in accordance with the purpose of measurement. Reagents for measuring various coagulation times are commercially available (for example, APTT reagent Coagpia APTT-N; manufactured by hydrographic medical Co., ltd.). The measurement of the coagulation reaction may be performed by any general means, for example, optical means for measuring the amount of scattered light, transmittance, absorbance, or the like, mechanical means for measuring the viscosity of plasma, or the like. The reaction start time of the coagulation reaction can be typically defined as a time when the coagulation reaction is started by mixing a reagent in a sample, and other times can be defined as reaction start times. The time for continuing the measurement of the coagulation reaction may be, for example, about several tens of seconds to 7 minutes from the time when the sample and the reagent are mixed. The measurement time may be a fixed value that is arbitrarily determined, but may be a time until the end of the detection of the coagulation reaction of each sample. Measurement of the progress of the coagulation reaction (photometry in the case of optical detection) can be repeated at predetermined intervals during the measurement time. For example, the measurement may be performed at intervals of 0.1 second. The temperature of the mixed solution in this measurement is a usual condition, and is, for example, 30 to 40 ℃ and preferably 35 to 39 ℃. In addition, various conditions for measurement can be set as appropriate depending on a sample to be measured, a reagent, a measurement means, and the like.

The series of operations in the measurement of the coagulation reaction described above may be performed using an automatic analysis device. An example of the automatic analyzer is an automatic blood coagulation analyzer CP3000 (manufactured by hydrops medical corporation). Alternatively, a part of the operation may be performed by a manual operation. For example, a sample to be measured can be prepared by a human, and the subsequent operation can be performed by an automatic analyzer.

2. Data parsing

2.1 Pre-processing and correction processing of data

Next, data analysis in step 2 will be described. Fig. 2 shows a flow of data analysis. The data analysis in step 2 may be performed simultaneously with the measurement of the coagulation reaction in step 1, or may be performed after the measurement of the coagulation reaction using data measured in advance. Preferably, the data analysis in step 2 is performed simultaneously with the measurement of the coagulation reaction in step 1, and the measurement of the coagulation reaction of the sample is terminated when data necessary for calculating the coagulation time of the sample to be measured can be acquired, and the process shifts to the measurement of the coagulation reaction of the subsequent sample.

In step 2a, measurement data in the measurement of the coagulation reaction is acquired. This data is data reflecting the progress of the coagulation reaction of the sample obtained in the APTT measurement in step 2 described above, for example. For example, data indicating a temporal change in the amount of progress (for example, the amount of scattered light) of a coagulation reaction after addition of a calcium chloride solution from a sample containing a sample to be measured and a reagent for measuring a coagulation time can be obtained. In the present specification, the data obtained by measuring the coagulation reaction is also referred to as coagulation reaction information.

Fig. 3 shows an example of the coagulation reaction information obtained in step 2 a. Fig. 3 is a coagulation reaction curve based on the amount of scattered light, the horizontal axis represents the elapsed time (coagulation reaction time) after the addition of the calcium chloride solution, and the vertical axis represents the amount of scattered light. Since time elapses and the coagulation reaction of the mixed liquid proceeds, the amount of scattered light increases. In the present specification, a curve showing such a change in the coagulation reaction amount with respect to the coagulation reaction time is referred to as a coagulation reaction curve.

The coagulation response curve based on the amount of scattered light shown in fig. 3 is generally S-shaped (\124716412514\1245289. On the other hand, the coagulation response curve based on the amount of transmitted light is generally reverse S-shaped (reverse 12471124644. In the following description, data analysis using a coagulation reaction curve based on the amount of scattered light as coagulation reaction information will be described.

If necessary, the coagulation reaction curve may be subjected to a pretreatment (step 2 b). The preprocessing may include smoothing for removing noise or zero point adjustment. Fig. 4 shows an example of the coagulation response curve of fig. 3 after pretreatment (smoothing treatment and zero point adjustment). Any known noise removal method can be used for the smoothing process. As shown in fig. 3, since the liquid mixture containing the sample to be measured scatters various lights, the amount of scattered light at the start of measurement (time 0) is greater than 0. By the zero point adjustment after the smoothing processing, as shown in fig. 4, the amount of scattered light at time 0 is adjusted to 0. FIGS. 5A and B show enlarged partial views of the coagulation response curve of FIG. 3 before and after pretreatment, respectively. In fig. 5B, smoothing processing and zero point adjustment are performed with respect to the data of fig. 5A.

The coagulation response curve is highly dependent on the fibrinogen concentration of the sample being tested. On the other hand, since the fibrinogen concentration varies among individuals, the height of the coagulation response curve varies depending on the sample to be measured. Therefore, in the present method, a correction process for relatively valuing the coagulation reaction curve after the pretreatment is performed in step 2c as necessary. According to this correction processing, a coagulation response curve independent of the fibrinogen concentration can be obtained, and thus the difference in the shape of the coagulation response curve after pretreatment between samples can be quantitatively compared.

In one embodiment, in the correction processing, the coagulation reaction curve after the pretreatment is corrected so that the maximum value becomes a predetermined value. Preferably, in the correction processing, a corrected coagulation reaction curve P (t) is obtained from the coagulation reaction curve after the pretreatment, based on the following formula (1). In the formula (1), D (t) represents the coagulation reaction curve after the pretreatment, dmax and Dmin represent the maximum value and the minimum value of D (t), respectively, drange represents the variation width of D (t) (i.e., dmax-Dmin), and a is an arbitrary value representing the maximum value of the corrected coagulation reaction curve.

P(t)＝[(D(t)-Dmin)/Drange]×A (1)

As an example, fig. 6 shows data corrected so that the coagulation reaction curve shown in fig. 4 becomes a maximum value 100. Note that, although the corrected value is corrected from 0 to 100 in fig. 6, other values (for example, from 0 to 10000, that is, a =10000 in the formula (1)) may be used. In addition, this correction process does not necessarily have to be performed.

Alternatively, the correction processing described above may be performed on a waveform relating to a coagulation speed described later or a parameter extracted from the waveform. For example, a waveform relating to the coagulation rate may be calculated for the coagulation reaction curve D (t) after the pretreatment without the correction treatment, and then converted into a value for P (t). Alternatively, after extracting a parameter from the waveform relating to the coagulation rate, the value of the parameter may be converted into a value corresponding to P (t).

In the present specification, the above-described corrected coagulation reaction curve and the coagulation reaction curve that has not been subjected to the correction processing are referred to as a corrected 0-order curve and an uncorrected 0-order curve, respectively, and they are collectively referred to as "0-order curve". In the present specification, the first order differential curves of the corrected 0 th order curve and the uncorrected 0 th order curve are referred to as a corrected first order curve and an uncorrected first order curve, respectively, and these are collectively referred to as "first order curves".

2.2 Calculation of waveform relating to coagulation speed

In step 2d, a waveform relating to the coagulation speed is calculated. In the present specification, the waveform relating to the coagulation speed includes an uncorrected first order curve and a corrected first order curve. The uncorrected first-order curve represents a value obtained by first-order differentiating the coagulation reaction curve (uncorrected 0-order curve), that is, a rate of change in the amount of coagulation reaction (coagulation rate) in an arbitrary coagulation reaction time. The corrected first-order curve indicates a value obtained by first-order differentiating the corrected coagulation reaction curve (corrected 0-order curve), that is, a relative change rate of the amount of the coagulation reaction in an arbitrary coagulation reaction time. Accordingly, the waveform relating to the coagulation speed may be a waveform indicating the coagulation speed of the coagulation reaction of the sample or a relative value thereof. In this specification, the value indicating the progress of blood coagulation including the coagulation rate and the relative value thereof indicated by the first order curve may be collectively referred to as a first order differential value. Differentiation of the coagulation response curve or the corrected coagulation response curve (uncorrected and corrected 0 th order curves) can be carried out using known methods. Fig. 7 shows a corrected first-order curve obtained by first-order differentiating the corrected 0-order curve shown in fig. 6. In FIG. 7, the horizontal axis represents the coagulation reaction time, and the vertical axis represents the first order differential value.

2.3 ) extraction of parameters

In step 2e, the extraction of the parameters of the waveform signature characteristic related to the coagulation speed is performed. More specifically, in the parameter extraction step, a predetermined calculation target region is extracted from the waveform relating to the coagulation speed, and then a weighted average time for the calculation target region is calculated. Based on the weighted average time, the blood coagulation time of the measured sample can be determined (step 3). This parameter is explained below.

First, a description will be given of a procedure for extracting a predetermined calculation target region from a waveform relating to a coagulation velocity. The calculation target region is a region in which the waveform of the waveform relating to the coagulation speed is equal to or greater than a predetermined lower limit value. More specifically, the calculation target region is a region (portion) of F (t) satisfying F (t) ≧ Vmax × x% when F (t) (t = time) is a waveform (first-order curve) relating to the coagulation speed and Vmax is a maximum value of F (t). More specifically, the calculation target region is a region (portion) of a first-order curve F (t) satisfying Vmax ≧ F (t) ≧ Vmax × x%. Therefore, "Vmax × x%" represents a lower limit value of the operation target region. The operation target region will be described with reference to fig. 8. The maximum Vmax of the first-order curves F (t) (t = time) and F (t) is shown in fig. 8. A base line indicating Vmax × x% is shown by a broken line, and times t1 and t2 when F (t) = Vmax × x%. The calculation target region is a region where F (t) is equal to or more than the baseline and Vmax is equal to or less than Vmax (F (t) ≥ Vmax × x%, t1 ≤ t ≤ t 2).

The weighted average point (vT, vH) corresponds to a "weighted average" of the calculation target region. The coagulation reaction time (t) at the weighted average point is set as a weighted average time vT. That is, the weighted average time vT is the time from the coagulation reaction start time to the weighted average point, and is the x-coordinate of the weighted average point. Further, vT is the center of gravity with respect to the horizontal axis of the operation target region. The weighted average height vH is the y-coordinate of the weighted average point.

In the present specification, the operation target region based on a predetermined lower limit value and vT and vH from the operation target region may be expressed based on a percentage of Vmax with respect to the lower limit value. For example, an operation target region in which x% of Vmax is a lower limit value may be referred to as an "x% operation target region", and vT and vH from the "x% operation target region" may be referred to as vTx% and vHx%, respectively. For example, an operand region having a lower limit of Vmax of 20% may be referred to as a 20% operand region, and vT of the operand region is represented by vT 20%. Fig. 8 shows weighted average points (vTx%, vHx%) of the calculation target region of F (t) having a lower limit value of x%.

The weighted average time vT and the weighted average height vH for the first order curve can be obtained in the following order. First, a data set in which the maximum value of the first-order curve F (t) is Vmax, the lower limit value of the calculation target area is x% of Vmax, and time t at which F (t) is equal to or greater than Vmax × x% is satisfied is t [ t1, \8230: ] t2] (t 1 < t 2). That is, F (t 1) = Vmax × x%, F (t 2) = Vmax × x%, and the data group from time t1 to time t2 is t [ t1, \8230, t2] (t 1 < t 2). At this time, the weighted average time vT and the weighted average height vH are calculated by the following expressions (2) and (3), respectively. From the determined vT and vH, a weighted average point (vTx%, vHx%) of the x% calculation target region is derived.

The lower limit value of the operation target region may be determined by a range greater than 0% of Vmax and smaller than Vmax. The calculation target region reflects the shape of the first-order curve, and the larger the lower limit value is, the more the calculation target region reflects the shape of the upper part of the first-order curve. In the method of the present invention, the lower limit value ("Vmax × x%") of the calculation target region for calculating the weighted average time used for measuring the coagulation time is preferably a predetermined value set in a range of 5 to 95% (i.e., x =5 to 95) of Vmax. More specifically, the lower limit of the calculation target region for calculating the weighted average time used for measuring the coagulation time is preferably a predetermined value set in the range of 5 to 95% (x =5 to 95) of Vmax, more preferably 5 to 50% (x =5 to 50) of Vmax, even more preferably 10 to 35% (x =10 to 35) of Vmax, when the coagulation time is APTT, and is preferably a predetermined value set in the range of 5 to 95% (x =5 to 95) of Vmax, more preferably 10 to 80% (x =10 to 80) of Vmax, even more preferably 25 to 50% (x =25 to 50) of Vmax, when the coagulation time is PT.

Fig. 9 shows the relationship between the calculation target region of the first-order curve and the calculated weighted average point. In fig. 9, the upper, middle, and lower stages show the calculation target region and the weighted average point (black circle) of the first-order curve when the lower limit value is 5%, 40%, and 75% of Vmax, respectively. As the calculation target region changes, the position of the weighted average point changes as shown in fig. 9.

In fig. 8 and 9, the calculation target region and the weighted average point thereof are described using the corrected first-order curve as an example, but the same parameters can be calculated even in the case of the uncorrected first-order curve.

3. Determination of the coagulation time

As shown in the examples described later, the weighted average time vT calculated in the above-described order has a high correlation with the blood coagulation time of the sample to be measured. Therefore, the coagulation time of the sample under test can be determined based on the weighted average time vT. For example, in the case where the coagulation time is APTT, PT, the weighted average time vT may be determined as the coagulation time. In the method of the present invention, by obtaining a weighted average of the first order curve of the coagulation reaction, the coagulation time can be measured with higher reliability without being affected by a multimodal peak due to measurement noise or coagulation abnormality, as compared with the case where the peak of the first order curve is simply detected.

In the method of the present invention, the coagulation time can be measured when the lower limit value (Vmax × x%) of the calculation target region is equal to or less than the maximum value Vmax of the first-order curve of the coagulation reaction. Therefore, the method of the present invention makes it possible to keep the coagulation reaction stable without the need of continuous measurement as in the conventional percentage method. Further, even when a plurality of samples are analyzed by an automatic analyzer, the method of the present invention does not require setting a long measurement time for an abnormal sample having a low coagulation ability as in the conventional percentage method. Therefore, according to the present invention, the analysis time of the sample can be shortened or optimized to improve the analysis efficiency.

4. Determination of coagulation factor concentration

Normal blood contains coagulation factors such as coagulation factors I to XIII, and abnormality or lack of these coagulation factors leads to abnormality of the coagulation ability. In general, the coagulation factor concentration of a sample to be measured can be measured based on the coagulation time of a measurement sample prepared from the sample to be measured using a reagent specific to the coagulation factor. Therefore, according to the method of the present invention, the coagulation factor concentration of the sample to be measured can be measured using the coagulation time of the measurement sample measured based on the weighted average time. In general, the coagulation factor concentration is measured based on a standard curve showing the relationship between the coagulation time and the coagulation factor concentration. Therefore, by applying the coagulation time of the measurement sample measured by the method of the present invention to a previously prepared calibration curve, the coagulation factor concentration of the sample can be measured. Preferred examples of the coagulation factor to be measured in the method of the present invention include factor I (fibrinogen), factor VIII, factor IX and the like.

In the method of the present invention, the lower limit value ("Vmax × x%") of the calculation target region for calculating the coagulation time (weighted average time) used for measuring the coagulation factor concentration is preferably a predetermined value set in a range of 5 to 95% (i.e., x =5 to 95) of Vmax, more preferably 30 to 95% (i.e., x =30 to 95) of Vmax, and still more preferably 60 to 75% (i.e., x =60 to 75) of Vmax.

5. Application to other measurement methods of coagulation reaction

The blood coagulation time measuring method of the present invention is described above by taking a case of measuring a coagulation reaction based on the amount of scattered light as an example. However, those skilled in the art can apply the method of the present invention to a blood coagulation time measuring method using other coagulation reaction measuring methods (for example, a blood coagulation reaction measuring method based on transmittance, absorbance, viscosity, and the like). For example, a first-order curve F (t) obtained from an inverse S-shaped coagulation reaction curve based on the transmitted light amount has opposite signs with respect to the scattered light amount. In such a case, it is obvious to those skilled in the art that the sign of F (t) is reversed in the calculation of the parameters, for example, the maximum value Vmax is replaced with the minimum value Vmin, the x% operand region is a region satisfying F (t) ≦ Vmin × x%, and the like.

Examples

The present invention will be described in further detail below with reference to examples, but the present invention is not limited to these examples.

Example 1 measurement of clotting time (APTT) based on weighted average time

1. Method for producing a composite material

1.1 Test specimen)

As the samples to be measured, a total of 24 samples of 9 samples of normal plasma and 15 samples of APTT-extended abnormal plasma were used. Normal Plasma used was Normal Donor Plasma manufactured by CliniSys Associates, ltd. As abnormal Plasma, 2 samples each were used as Plasma lacking coagulation factors (Factor Deficient Plasma), plasma lacking Factor viii, plasma lacking Factor FIX, plasma lacking Factor FXI, and Plasma lacking Factor FXII, 2 samples were used as Lupus Anticoagulant positive Plasma (Lupus Anticoagulant Plasma), and 5 samples were used as unfractionated heparin-containing Plasma (Anticoagulant Plasma) (both CliniSys Associates, ltd.).

1.2 Reagent)

Coagpia APTT-N (manufactured by hydropsy medical Co., ltd.) was used as the APTT reagent.

1.3 Measurement of coagulation reaction

The measurement of the coagulation reaction was performed using a blood coagulation autoanalyzer CP3000 (manufactured by hydrographic medical Co., ltd.). After 50. Mu.L of the sample was dispensed into a cuvette (reaction vessel), the cuvette was heated at 37 ℃ for 45 seconds, 50. Mu.L of an APTT reagent heated to about 37 ℃ was added to the cuvette, and after 171 seconds, 50. Mu.L of a calcium chloride solution was added to the cuvette to initiate the coagulation reaction. The reaction was carried out while maintaining at about 37 ℃. Measurement of the coagulation reaction (photometry) was performed by irradiating a cuvette with light from an LED light having a wavelength of 660nm, and measuring the amount of scattered light of the 90-degree side-scattered light at 0.1-second intervals. The maximum measurement time was 360 seconds (3600 data, 0.1 second interval).

1.4 APTT determination (percentage method)

The APTT of each sample was determined by the percentage method. That is, within the measurement time, the time at which the amount of scattered light reaches the maximum is determined as the solidification reaction end point, and the time at which 50% of the amount of scattered light at the solidification reaction end point is determined as the APTT. The kinds and numbers of the samples to be measured and the minimum and maximum values of APTT in the samples of each kind are shown in table 1.

[ Table 1]

1.5 Preparation of a solidification reaction Curve

After smoothing processing including noise removal is performed on photometric data from each sample, zero point adjustment processing is performed so that the amount of scattered light at the photometric start time becomes 0, and a coagulation response curve P (t) is calculated. Next, the coagulation reaction curve is corrected so that the maximum value Pmax of the coagulation reaction curve becomes 100, and the corrected coagulation reaction curve (corrected 0-order curve) obtained is first-order differentiated to calculate a corrected first-order curve.

1.6 Calculation of a weighted average time vT

From the first-order curves obtained from the samples, weighted average times (vT 5% to vT 95%) for 5% to 95% of the calculation target region were calculated. The lower limit value x% of the calculation target region is set to 19 stages of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, and 95% of the maximum height Vmax (100%) of the first-order curve F (t), and times t1 and t2 satisfying F (t) = Vmax × x% are obtained, respectively (t 1 < t 2). Using the above equation (2), 19 weighted average times vT5% to vT95% were calculated from 24 samples each.

2. APTT determination based on weighted average time

2.1 Correlation of weighted average time to APTT

The correlation of APTT to weighted average time based on the percentage method was evaluated. For each weighted average time (vT 5% to vT 95%) for 5 to 95% of the calculation target region, linear regression analysis based on APTT with the percentage method was performed to determine the slope, intercept, and correlation coefficient of the regression line.

Fig. 10 shows a linear regression line of vT20% in the 20% operand region with respect to the APTT based on the percentage method for 24 samples. vT20% has a high correlation for APTT based on the percentile method. Fig. 11 shows the slope, intercept, and correlation coefficient of the linear regression line of vT (vT 5% to vT 95%) in 5 to 95% of the calculation target region with respect to the percentage APTT. In the region of 5 to 95% of the calculation target region, the inclination of the regression line is 0.95 to 1.01, the intercept is-2.0 to 0.4, and the correlation coefficient is 0.998 to 1.000 (fig. 11A to C). The APTT measurement method based on the weighted average time was confirmed to have the performance of satisfying the in vitro diagnostic medical approval criterion "in terms of correlation coefficient of 0.9 or more and slope of regression straight line of 0.9 to 1.1 as compared with the control measurement method" in japan, respectively, in the total of 5% to 95% of the target region. These results indicate that APTT can be determined based on vT.

2.2 Accuracy of assay

The weighted average time in 5 to 95% of the calculation target area for 24 samples was compared with the control (APTT based on the percentage method). Each row of the table in fig. 12AB indicates the weighted average time (vT 5% to vT 95%) of each sample, and is indicated by gray when the difference between the weighted average time and the control falls within ± 5% of the control (fig. 12A) and within ± 2.5% (fig. 12B). In 5 to 50% of the calculation target area, the weighted average time of all samples matches the control with an error within ± 5%, and in 10 to 35% of the calculation target area, the weighted average time of all samples matches the control with an error within ± 2.5%.

Example 2 coagulation time (PT) determination based on weighted average time

1. Method of producing a composite material

1.1 Test sample)

As the test samples, a total of 23 samples of 9 samples of normal plasma and 14 samples of PT-extended abnormal plasma were used. Normal plasma healthy human plasma was used. Abnormal plasma the plasma of patients administered with warfarin anticoagulated with an anticoagulant was used, 5 samples having PT-INR values of 1 to 2, 5 samples of 2 to 3, and 4 samples of 3 to 4, which represent the concentration of warfarin in blood, were used.

1.2 Measurement of coagulation reaction

The measurement of the coagulation reaction was performed using a blood coagulation automatic analyzer CP3000 (manufactured by hydrops medical Co., ltd.). After 50. Mu.L of the sample was dispensed into a cuvette (reaction vessel), the cuvette was heated at 37 ℃ for 45 seconds, and then 100. Mu.L of a thromboplastin solution heated to about 37 ℃ was added to the cuvette to initiate the coagulation reaction. The reaction was carried out while maintaining at about 37 ℃. Measurement of coagulation reaction (photometry) was performed by irradiating a cuvette with light from an LED light having a wavelength of 660nm, and measuring the amount of scattered light of 90-degree side-scattered light at 0.1-second intervals. The maximum measurement time was 300 seconds (data number 3000, 0.1 second interval).

1.3 PT determination (percentage method)

The PT of each sample was determined by the percentage method. Within the measurement time, the time when the amount of scattered light reached the maximum was determined as the end point of the solidification reaction, and the time when 45% of the amount of scattered light reached the end point of the solidification reaction was determined as PT. The types and numbers of the samples to be measured and the minimum and maximum values of PT in the samples of each type are shown in table 2.

[ Table 2]

1.4 Computation of weighted average time vT)

Using the same procedure as in 1.5) and 1.6) of example 1, calibration first-order curves for 23 samples were calculated, and weighted average times vT5% to vT95% were calculated.

2. Weighted average time based PT determination

2.1 Correlation of weighted average time with PT

The slope, intercept and correlation coefficient of the regression line were obtained by performing linear regression analysis with PT measured by the percentage method for each weighted average time (vT 5% to vT 95%) of 5 to 95% of the calculation target region in the same manner as in 2.1) of example 1.

Fig. 13 shows linear regression lines of vT30% of the 30% operation target region with respect to PT by the percentage method for 23 samples. vT30% has a high correlation with PT based on the percentage method. Fig. 14 shows the slope, intercept, and correlation coefficient of the linear regression line of vT (vT 5% to vT 95%) in the 5 to 95% calculation target region with respect to the percentage PT. In the period of 5 to 95% of the calculation target region, the inclination of the regression line was 0.94 to 1.05, the intercept was-0.1 to 0.7, and the correlation coefficients were all 1.000 (fig. 14A to C). It was confirmed that the PT measurement method based on the weighted average time has performance satisfying the in vitro diagnostic drug approval standard "in japan's ministry of health, life and labor" that the correlation coefficient is 0.9 or more and the inclination of the regression straight line is 0.9 to 1.1 as compared with the control measurement method, in all conditions from 5% to 95% of the calculation target region. These results indicate that PT can be measured based on vT.

2.2 Accuracy of assay

The weighted average time for 5 to 95% of the calculation target region for 23 samples was compared with the control (PT by percentage method). Each row of the table in fig. 15AB indicates the weighted average time (vT 5% to vT 95%) of each sample, and is indicated in gray when the difference between the weighted average time and the control falls within ± 5% of the control (fig. 15A) and within ± 2.5% (fig. 15B). In 10 to 80% of the calculation target area, the weighted average time of all samples matches the control with an error within ± 5%, and in 25 to 50% of the calculation target area, the weighted average time of all samples matches the control with an error within ± 2.5%.

Example 3 fibrinogen concentration determination based on weighted average time

1. Method for producing a composite material

1.1 Test specimen)

Human Fibrinogen (Human Fibrinogen, product name: FIB 2, manufactured by Enzyme Research Laboratories) was added to Human Fibrinogen-free Plasma (Fibrinogen, product name: fg, manufactured by Affinity Biologicals Inc.) to prepare a sample (sample 10) having a Fibrinogen concentration ([ Fbg ]) of 980 mg/dL. As a standard sample for preparing a standard curve, sample 10 and physiological saline were mixed at volume ratios of 1: 9, 7: 3 and 10: 0 to prepare three samples having fibrinogen concentrations ([ Fbg ") of 98mg/dL, 686mg/dL and 980mg/dL, respectively. In addition, 10 samples of concentration series having different fibrinogen concentrations ([ Fbg ]) in stages were prepared by mixing the sample 10 and the human fibrinogen-removed plasma at a volume ratio of 1: 9 to 10: 0 (Table 3).

[ Table 3]

Sample(s)	Dilution ratio	[Fbg](mg/dL)
			1	1∶9	98
2	2∶8	196
			3	3∶7	294
4	4∶6	392
			5	5∶5	490
6	6∶4	588
			7	7∶3	686
8	8∶2	784
			9	9∶1	882
10	10∶0	980

1.2 Measurement of coagulation reaction

As the fibrinogen measurement reagent, a thrombin reagent attached to CoagpiaFbg (manufactured by hydropneumatic Co., ltd.) and a sample diluent were used. The measurement of the coagulation reaction was performed using a blood coagulation automatic analyzer CP3000 (manufactured by hydrops medical Co., ltd.). mu.L of the sample and 90. Mu.L of the sample dilution were dispensed into a cuvette, and after heating at 37 ℃ for 45 seconds, 50. Mu.L of a thrombin reagent heated to about 37 ℃ was added to the cuvette to start the coagulation reaction. The reaction was carried out while maintaining at about 37 ℃. The measurement of the coagulation reaction was performed by irradiating a cuvette with light having a wavelength of 660nm as a light source and measuring the amount of scattered light of 90-degree side scattered light at 0.1 second intervals. The maximum measurement time was 300 seconds (data number 3000, 0.1 second interval). The measurement of the coagulation reaction was performed 2 times for each of 3 standard samples and 10 concentration series samples.

1.3 Calculation of fibrinogen concentration based on coagulation time measurement (percentage method)

The clotting times of the 3 standard samples and the 10 samples of the concentration series were determined by the percentage method. That is, the time at which 63% of the amount of scattered light at the end of the solidification reaction is reached is determined as the solidification time. The clotting time of each sample was determined 2 times based on 2 clotting reaction measurements. For the coagulation time measured by 3 standard samples, the average value of 2 measurements was calculated, and the logarithm of the average value was plotted against the logarithm of [ Fbg ] (mg/dL) of the standard sample to prepare a standard curve by the percentage method. From the prepared calibration curve, the fibrinogen concentration (calculated value of [ Fbg ] by percentage method, mg/dL) of each concentration series sample was calculated.

1.4 Calculation of fibrinogen concentration based on the weighted mean time vT

The weighted average time vT5% to vT95% was calculated for each of the 2 concentration series samples in the same order as in 1.5) and 1.6) of example 1, and data of the weighted average time of 20 sample components was obtained. For 3 standard samples, 2 weighted average times vT5% to vT95% were calculated, and the average of 2 measurements was calculated. The logarithm of the average value is plotted against the logarithm of [ Fbg ] (mg/dL) of the standard sample to form a standard curve based on the weighted average time. From the prepared calibration curve, the fibrinogen concentration (calculated value of [ Fbg ] based on the weighted average time, mg/dL) of each concentration series sample was calculated.

2. Evaluation of fibrinogen concentration determination based on weighted average time

2.1 Correlation resolution)

Fig. 16 shows a linear regression line of the [ Fbg ] calculated value (mg/dL) based on the weighted average time calculated from vT35% of the 35% calculation target region with respect to the [ Fbg ] calculated value based on the percentage method based on the data (n =10 × 2) of the concentration series samples. The [ Fbg ] operation value based on vT35% has a high correlation with the [ Fbg ] operation value based on the percentage method. Fig. 17 shows the slope, intercept, and correlation coefficient of the linear regression line of the [ Fbg ] calculated value from vT (vT 5% to vT 95%) of the 5 to 95% calculation target region with respect to the [ Fbg ] calculated value by the percentage method. In the 5 to 95% calculation target region, the gradient of the regression line is 0.94 to 1.02, the intercept is-31.5 to 51.0, and the correlation coefficient is 0.990 to 0.996 (fig. 17A to C). Since the fibrinogen concentration measurement method based on the weighted average time brings about the same result as the method based on the standard of the percentage method under all the conditions from 5% to 95% of the region to be calculated, it was confirmed that the in vitro diagnostic medical product acceptance criterion "satisfying japanese heighobian labour and labour province" had the performance of having a correlation coefficient of 0.9 or more compared with the control measurement method and a gradient of 0.9 to 1.1 in the regression straight line formula ". These results indicate that fibrinogen concentration can be determined based on vT.

2.2 Accuracy of the assay

The calculated value of [ Fbg ] based on the weighted average time of 5 to 95% of the calculation target region in the data of the density series samples (10 samples × 2 in table 3) was compared with the expected value (Fbg density of the sample shown in table 3). Each row of the table in fig. 18AB indicates the weighted average time (vT 5% to vT 95%) of each sample, and is indicated by gray when the error between the [ Fbg ] calculated value and the expected value based on the weighted average time falls within ± 10% (fig. 18A) and ± 5% (fig. 18B). In the 30-95% calculation target region, the error of the [ Fbg ] calculation value of all samples is within + -10%, and in the 60-75% calculation target region, the error of the [ Fbg ] calculation value of all samples is within + -5%.

Claims

1. A blood coagulation time measuring method comprising:

measuring a coagulation reaction with respect to a sample obtained by mixing a sample to be measured and a reagent for measuring a coagulation time,

2. The method of claim 1, wherein the coagulation speed-related waveform is a coagulation response curve or a first order differential curve of relative values thereof.

3. The method according to claim 1 or 2, wherein when the waveform relating to the coagulation speed is set to F (t), and times when F (t) is x% of a maximum value are set to t1, t2, the weighted average time is represented by the following equation:

wherein t is time, x is a predetermined value set in the range of 5 to 95, and t1 < t2.

4. The method according to any one of claims 1 to 3, wherein the sample to be tested is plasma.

5. The method according to any one of claims 1 to 4, wherein the blood coagulation time is activated partial thromboplastin time APTT, prothrombin time PT, coagulation time in fibrinogen concentration assay.

6. A method for measuring a coagulation factor concentration, comprising measuring the coagulation factor concentration of a sample to be measured based on the blood coagulation time of the sample measured by the method according to any one of claims 1 to 5.

7. The method of claim 6, wherein the sample being tested is plasma.

8. The method of claim 6 or 7, wherein the coagulation factor is fibrinogen.

9. The method of claim 8, wherein the blood clotting time is the clotting time in a fibrinogen concentration assay.